ID2 and HIF-1α collaborate to protect quiescent hematopoietic stem cells from activation, differentiation, and exhaustion

Defining mechanism(s) that maintain tissue stem quiescence is important for improving tissue regeneration, cell therapies, aging, and cancer. We report here that genetic ablation of Id2 in adult hematopoietic stem cells (HSCs) promotes increased HSC activation and differentiation, which results in HSC exhaustion and bone marrow failure over time. Id2Δ/Δ HSCs showed increased cycling, ROS production, mitochondrial activation, ATP production, and DNA damage compared with Id2+/+ HSCs, supporting the conclusion that Id2Δ/Δ HSCs are less quiescent. Mechanistically, HIF-1α expression was decreased in Id2Δ/Δ HSCs, and stabilization of HIF-1α in Id2Δ/Δ HSCs restored HSC quiescence and rescued HSC exhaustion. Inhibitor of DNA binding 2 (ID2) promoted HIF-1α expression by binding to the von Hippel-Lindau (VHL) protein and interfering with proteasomal degradation of HIF-1α. HIF-1α promoted Id2 expression and enforced a positive feedback loop between ID2 and HIF-1α to maintain HSC quiescence. Thus, sustained ID2 expression could protect HSCs during stress and improve HSC expansion for gene editing and cell therapies.

A, Total HSCs following indicated treatments of Id2 +/+ HSCs in stem cell expansion assays as described in Figure 6A. B, Total HSCs following the indicated treatments of Id2 +/+ HSCs in maintenance cultures as described in Figure 7A. unpaired Students t test, and a two-way ANOVA with Dunnet's correction was used for multiple means testing in A, and a one-way ANOVA with Dunnet's correction was used for multiple means testing in J. *P ≤ 0.05, and **P ≤ 0.01, ***P ≤ 0.001. NS, not significant.

Cell Lines
EML cells were maintained in Iscove's modified eagle medium 20% horse serum (Gem Cell, 100-508), 1 % penicillin/streptomycin (Gibco, 15140122), and BHK conditioned medium containing SCF at 37 o C, 5% CO2 as described previously (1)  Gy irradiated recipient mice. Six weeks after BMT, mice were treated with two doses of pIpC as above. Four weeks after pIpC treatment, mice were treated with four doses of 135 mg/Kg 5flurouracil (5-FU) (NIH pharmacy) at one-week intervals. Survival of mice was monitored following the first 5-FU injection. Rough and lethargic mice were euthanized in accordance with the "Guide for the Care and Use of Laboratory Animals."
All cytokines were purchased from Peprotech Inc. In vitro assays were performed using BMCs from at least three different mice for each group. BMCs were cultured for 48 hrs and HSPCs were analyzed for ID2/eYFP expression by flow cytometry. Alternatively, Lincells were cultured in StemSpan containing msSCF, huTPO and either 1 nM, 10 nM, or 100 nM echinomycin (SML0477, Sigma). HSPC analysis was performed 24 and 48 hours after culturing.

Plasmid Constructs
TAAGGATCCTGCCCGGATCCTCACAGAT followed by digestion with BAMHI and NHEI digestion and standard cloning procedures. ID2promoterpGL4.1 was described previously (2).
To calculate viral titers, NIH3T3 cells were infected with serial dilutions of viral supernatants.
For knockdown, Lin-cells were transduced with shRNA lentivirus by spinoculation. Briefly, Lin-cells were isolated from Mx1-Cre; Id2 Fl/Fl mice 2 weeks after administration of pIpC using immune-magnetic bead separation. Lin-cells were cultured in Stemspan medium containing msSCF (100 ng/mL), huTPO (100 ng/mL), msFGF1 (10 ng/mL), huIGF2 (20 ng/mL), and Angiopoietin 2 (50 ng/mL) at 5 × 10 5 cells/0.5mL for 12 hours. Twelve hours after culture, the cells were subjected to first round of shRNA mediated lentiviral transduction, where lineage depleted cells were spun at 2000 x g for 90 minutes at 37°C. The cells were then washed and reseeded with fresh complete medium. After 24 hours, a second round of transduction was performed as described above, and the cells were cultured in puromycin (5 μg/mL). Ninety-six hours after the second lentiviral mediated transduction, the Lincells were harvested for qRT-PCR or for HSPC analysis and HIF-1α expression by flow cytometry.

Co-Immunoprecipitation Assays
Protein lysates were harvested in low salt lysis/binding buffer containing protease inhibitor cocktail (Roche) and phosphatase inhibitors (Active Motif Quantification was carried out with RSEM using the transcriptome bam file created by STAR. Differentially expressed genes (DEGs) were analyzed with Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, Inc., Redwood City, CA, USA) and Gene Set Enrichment Analysis (GSEA, Broad Institute, Cambridge, MA, USA). For IPA, pathways above a log Pvalue of 1.33 and a Z-score above 1 were considered statistically significant. For GSEA pathways, Normalized enrichment scores ≥ 1.3 and a FWER p-value below 0.05 were considered significant.

Single Cell RNA-Seq Analysis
Approximately 1,000 HSCs (Lin -Sca-1 + , Kit + , FLT3 -, CD150 + , CD48 -) were sorted from Mx1-Cre; Id2 +/+ or Mx1-Cre; Id2 Fl/Fl mice and captured using the 10X Genomics Chromium, as previously described (http://software.10xgenomics.com/single-cell). The number of cells captured ranges from 384 to 686 and mean reads per cell ranged from 218,801 to 550,748. Cells with extremely low number of UMI counts were filtered out. Median genes found per cell ranges from 2,687 to 3,274 and the total number of genes detected ranges from 14,556 to 15,256. All metrics are within the expected range for 10x Chromium single cell libraries. The single-cell barcoded cDNA libraries were sequenced on NextSeq500 (Illumina) for two runs. The sequencing run was setup as a 26 cycles + 57 cycles in a non-symmetric run. The 10X Genomics Cellranger toolkit (v2.2.0) was used to perform de-multiplexing, allowing 1 mismatch in the barcodes, and alignment to the mm10 transcriptome and gene-barcode matrices. Pre-processing, removal of highly variable genes and contaminating immune cells, dimensionality reduction, and clustering analyses procedures were applied to each data set and merged data sets using the Seurat R package (Satija 2015, Nat. Biotech.) https://satijalab.org/seurat/v3.0/pbmc3k_tutorial.html). In brief, cells that had feature counts